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. 2017 Jul 19;8:15878. doi: 10.1038/ncomms15878

Figure 3. TRPV1-expressing hippocampal neurons are positive for OLM neuron markers and NGF upregulates TRPV1.

Figure 3

(a) Immunostains of hippocampal cultures for TRPV1 and Somatostatin (SOM), reelin, GAD65, VIP or Parvalbumin (PV). (b) Immunostain of hippocampal neuron culture for TRPV1, mGluR7 and MAP2; TRPV1-expressing neurons high mGluR7-positive innervation compared to surrounding neurons. (c) Quantitation of co-localization of immunofluorescence signal of different markers with TRPV1 neurons in hippocampal cultures. VR.5′ sv quantification indicates signal from the C-terminal TRPV1 antibody, compared to the N-terminal TRPV1 antibody (Fig. 1e). Cells/images analysed: TRPV1/VR.5′ sv n=54, TRPV1/mGluR7 n=22, TRPV1/somatostatin n=62, TRPV1/reelin n=52, TRPV1/GAD65 n=60, TRPV1/PV n=26, TRPV1/VIP n=22, from 3 to 6 cultures; scale bar, 20 μm. (d) Immunostain (left) and quantitation (right) of hippocampal cultures showing that NGF is highly expressed in TRPV1-expressing hippocampal neurons, compared to surrounding neurons (n=15 images from three cultures; error=s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, **P<0.01). (e) Images of control hippocampal neuron cultures and cultures treated with 1 ng ml−1 NGF at DIV12–13 for 21–24 h, immunostained for TRPV1 (left). Quantitation of TRPV1 immunofluorescence intensity normalized to control conditions is shown on the right (n=26 (control), and 12 (NGF) from nine cultures; error=s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, ***P<0.001). (f) Western blot of TRPV1 in three repetitions of treatment of cultured hippocampal neurons with NGF compared to control. The levels of synaptobrevin-2 (used as a negative control) are unchanged. Tubulin serves as a load control. (g) Immunostain of a hippocampal section with TRPV1, vGluT1 and DAPI, showing high vGluT1 innervation of a TRPV1-expressing neuron in the stratum oriens; scale bar=20 μm. (h) Immunostain of a dissociated hippocampal neuron culture with TRPV1, vGluT1 and MAP2 (to mark neuronal cell bodies and processes), showing high vGluT1 innervation of a TRPV1-expressing neuron; scale bar, 20 μm.